Project Summary/Abstract The mitochondrial calcium uniporter (the uniporter) is a multi-subunit Ca2+ ion channel that imports cytoplasmic Ca2+ into the mitochondrial matrix. In mammalian cells, the uniporter plays a crucial role in regulating ATP generation, buffering intracellular Ca2+, and modulating cell-death pathways. Its dysfunction has been implicated in a wide range of pathological conditions, including a human neuromuscular disorder characterized by proximal myopathy and learning difficulties. This project seeks to expand the knowledge base in the molecular mechanisms underlying the uniporter's key roles in pathophysiology. Specific aims include developing new electrophysiological tools, and using established methods to address fundamental questions in ion transport and gating. Currently, mechanistic studies of the uniporter have been impeded by a technical barrier: The small size of mitochondria makes it difficult to apply patch-clamp electrophysiology to analyze the channel in native environments. In Aim #1, we solved this problem by targeting uniporter proteins to alternative membrane systems, including reconstituted phospholipid bilayers and cell plasma membranes. Both systems offer much straightforward electrophysiological access for high-resolution recordings in macroscopic and single-channel levels. We plan to fully establish these tools so that researchers can begin to adopt classical ion-channel electrophysiology to illuminate most fundamental mechanisms of the uniporter. While developing new techniques, we will also use a CRISPR-based strategy already in use in my lab to attack key mechanistic questions. (1) How does a regulatory MICU1 subunit inactivate the uniporter in resting cellular conditions (Aim #2)? (2) How do MCU and EMRE, the membrane-embedded subunits of the uniporter, form an open Ca2+ pathway for Ca2+ to permeate mitochondrial membranes (Aim #3)? Several results, including a mutation that unexpectedly abolishes uniporter inactivation by MICU1, and the discovery of a unique MCU chimera that can conduct Ca2+ without EMRE present, allow us to formulate logical and testable hypotheses to answer these important but also difficult questions. Completion of this project can improve the scientific knowledge necessary to design new therapies to treat disease by modulating mitochondrial Ca2+ homeostasis. Moreover, human uniporter proteins purified here can be used for high-throughput screening assays to identify uniporter-targeting pharmacological compounds. New electrophysiological methods will allow detailed analysis of drug kinetics, required to improve lead compounds for potential therapeutic use.